Anal. Chem. 1006,67, 962-968
Supercritical Fluid Chromatographic Separation of /?=Blockers on Chyrosine=A: Investigation of the Chiral Recognition Mechanism Using Molecular Modeling N. Bargmann-Leyder, C. Sella, D. Bauer, A. Tambut6,t and M. Caude* Laboratoire de Chimie Analytique de /‘€cole Superieure de Physique et Chimie lndustrielles de Paris,# 70 rue Vauquelin, 75231 Paris cedex 05, France
Using supercriticalfluid chromatography(SFC),the direct separation of a series of p-blockers has been carried out on the commercially available ChyRoSine-A and on its improved version. Surprisingly, these solutes appeared to be poorly resolved using normal phase liquid chromatography (NPLC). The chromatographicbehaviors (both in SFC and NPLC) of various analogues of propranolol have been studied, and further spectroscopic investigations have been carried out, Starting from these data, a detailed chiral recognition mechanism has been elaborated based on molecular modeling. It appeared that the solvating effect of carbon dioxide induces a change of the propranolol conformations that is geometrically favorable to the chiral discrimination, this change of conformation occurs in the presence of carbon dioxide only if the solute bears both an amino proton and an ether function separated the one from the other by three carbon atoms. It is widely recognized that P-adrenergic blocking agents @?blockers), widely used in the treatment of angina pectoris, hypertension, cardiac arrhythmias, and anxiety exhibit drastically different pharmacologicalpotencies and actions according to their nature and stereogenic form.’ Accordingly, chromatographic methods for separating ,&blocker enantiomers (in order to ascertain their stereochemical purity) have been extensively developed.2 Although it is possible to converse P-blockers into diastereoisomers that can be resolved on an achiral column, the method consisting of use of chiral stationary phases (CSPs) is often preferred for accuracy reasons. Four types of CSPs have been successfully used for the direct resolution of P-blockers: brushtype CSPS,~,~ cyclodextrin CSPS,~CSPs derived from cellul0se,6~~ + Present address: Direction des Recherches et Etudes Techniques, Centre d’Etudes du Bouchet, BP No. 3, Le Bouchet, 91710 Vert-le-Petit, France. Unite de recherche No. 437 associke au CNRS. (1) k e n s , E. J. Racemates-an impediment in the use of agrochemicals. In Chiral Separations by HPLC: Applications to Pharmaceutical Compounds, Wiley/Ellis Honvood: Chichester, UK, 1989, pp 31-68. (2) Lough,W., Ed. Chiral Liquid Chromatography; Blackie: London, 1989. (3) HPLCApplication Data ojSumichira1 OA; Sumika Chemical Analysis Service Ltd., Osaka, Japan. (4) Pirkle, W. H.; Burke, J. A,, 111J Chromatogr. 1991,557,173-185. (5) h s t r o n g , D. W.; Chen, S.; Chang, C.; Chang, S. J. Liq. Chromatogr. 1992, 15 (3), 545-556. (6) Okamoto, Y.; Kawashima, M.; Aburatani, R.; Hatada, IC; Nishiyama, T.; Masuda, M. Chem. Lett. 1986,1237-1240. (7) Krstulovic, A M.; Fouchet, M. H.; Burke, J. T.; Gillet, G.; Durand, A J. Chromatogr. 1988,452,477-483.
*
952 Analytical Chemistry, Vol. 67, No. 5, March 7, 7995
and protein-based CSPS.~-” The choice of a class I (brush-type) CSP for the separation of B-blocker enantiomers turns out to be an interesting alternative owing to their low price, high efficiency, separative potentiel, and good stability. Among class I CSPs, a CSP derived from (R)-N-(3,5dinitrobenzoyl)phenylglycine (DNBPG) was the first employed for the resolution of P-blockers. Nevertheless, the prior derivation of the amine function under amide, urea, or carbamate was required in order to reduce its basicity and shorten elution times. In that way, Pirkle et a1.12first resolved propranolol as lauryl amide derivative, Wainer et assayed propranolol enantiomers (as oxazolidin-2-onederivatives) in human serum and Yang et al.14 carried out the enantiomeric separation of seven ,!%aminoalcohols as a-naphthylurea derivatives. Other class I CSPs have been used for the chromatographic resolution of B-blockers: NJVI-(3,5dinitrobenzoyl)-truns-1,2-dt aminocyclohexaneCSP (DACH-DNB) (as oxazolidin-2-onederivat i v e ~ ) and ’ ~ Sumichiral-OA 4100 and 4600 CSPs (without derivat i ~ n ) We . ~ note that Ohwa et a1.I6prepared a CSP derived from proline for the direct resolution of P-blockers, but rather low resolutions were achieved. Recently, Pirkle and Burke4proposed a CSP derived from N-(3,5dinitrobenzoyl)-a-amino-2,2-dimethyl4pentyl phosphonate (commercialized under the trade name a-Burke 1) for the direct resolution of P-blockers. Two years ago, we demonstrated for the first time the ability of tyrosine-derived CSPs (brush-type CSPs) to separate ,&blocker enantiomers without any derivation prior inje~tion.’~Using supercritical fluid chromatography (SFC) , the direct separation of a series of P-blockers has been carried out on the commercially available ChyRoSine-A and on its improved version (Figure 1): facile separations have been achieved (1.P R, < 7) within short analysis times, the mobile phase consisting of a mixture of carbon dioxide-methanol-n-propylamine. Surprisingly, these solutes are poorly resolved using normal phase liquid chromatography (8) Hermansson, J. J. Chromatogr. 1985,325,379-384. (9) Enquist, M.; Hermansson, J. Chirality 1989,1, 209-215. (10) Kusters, E.; Giron, D. HRC CC, J. High Resolut. Chromatogr. Chromatogr. Commun. 1986,9,531-533. (11) Haginaka, J.; Wakai, J.; Takahashi, IC; Yasuda, H.; Katagi, T. Chromatographia 1990,29,587-592. (12) Pirkle, W. H.; Finn, J. M.; Schreiner, J. L.; Hamper, B. C.J. Am. Chem. Soc. 1981,103,3964-3966. (13) Wainer, I. W.; Doyle, T. D.; Donn, IC H.; Powell, J. R. J. Chromatogr, 1984, 306,405-411. (14) Yang, 8.; Sun, Z. P.; Ling, D. K J. Chromatogr. 1988,447,208-211. (15) Gasparrini, F.;Misiti. D.; Villani. C.; LaTorre, F.J. Chromatogr. 1991,539, 25-36. (16) Ohwa, M.; Akiyoshi, M.; Mitamura, S.J. Chromatogr. 1990,521, 122-127. (17) Siret, L.; Tambute, A.; Caude, M.; Rosset, R Chirality 1992,4,36-42. 0 1995 American Chemical Society 0003-2700/95/0367-0952$9.00/0
H I
CSP 1: R=n-Butyl (ChyRoSine-A) CSP 2:R=rerf-Butyl Figure I. Structures of the
chiral stationary phases (CSPs).
(NPLC). The specific solvation of 1,2-amino alcohols by carbon dioxide was evidenced by NMR experiments, and it appeared that carbon dioxide acts as an in situ complexing agent toward the amino alcohol by setting-up of a bridge between the hydroxyl and the amine protons of the solute.18 In that way, we demonstrated that the resulting complex possesses lower acid-basic properties and higher conformational rigidity, responsible for chiral discrimination. Further investigations have been carried out in our laboratories in order to get better insights into this intricate phenomenon. In that way, the chromatographic behaviors (both in SFC and in NPLC) of various analogues of propranolol have been studied and further spectroscopic investigations (NMR, infrared spectroscopy) have been carried out. Starting from these data, a detailed chiral recognition mechanism has been elaborated based on molecular modeling. In fact, molecular modeling has been proven to be a powerful technique to design chemical reactions or liquid-liquid extraction^^^-^^ as well as to design CSPs and solutes with a view to proposing chiral recognition mechanism^.^^-^^ EXPERIMENTAL SECTION
Apparatus. 'H NMR spectra were recorded at 360 MHz on a Bruker 360 AMX spectrometer or at 200 MHz on a Bruker WP 200 spectrometer, at 296 K, using tetramethylsilane (TMS) as internal standard and [2Hlchloroformor [2H41methanolas solvent. Analytical liquid chromatography was performed with a modular liquid chromatograph Gilson (Villiers-le-Bel, France) equipped with a Model 303 pump, a Model 802 manometric module, a Model 811 dynamic mixer (1.5 mL), and a W-116 variablewavelength detector (when carbon disulfide was added to the mobile phase, a differential refractometer detector, Waters R401, was used). All results were recorded with a Shimadzu C-WA integrator vouzart et Matignon, Vitry-sur-Seine, France). The standard operating conditions were flow rate 2 mL/min and room temperature. Analytical supercritical fluid chromatography was performed with a modular supercritical chromatograph (SF3) (Gilson). Carbon dioxide (kept in a container with an eductor tube) was passed into a Model 308 pump through an ethanol cooling bath. (18) Siret, L.; Bargmann, L.; Tambute, A,; Caude, M. Chirality 1992,4, 252262.
Sella, C.; Bauer, D. Solvent Extr. Ion. Exch. 1992,10 (3), 491-507. Sella, C.; Bauer, D. Solvent Eztr. Ion. Exch. 1992,10 (4), 579-599. Sella, C.; Bauer, D. Solvent Extr. Ion. Exch. 1993,12 (3), 395-410. Topiol, S.; Sabio, M.J. Chromatogr. 1989,461, 129-137. Sabio, M.; Topiol, S. Chirality 1991,3,56-66. Hanai, T.; Hatano, H.; Nimura, N.; Kinoshita, T. 1.Liq. Chromatogr. 1993, 16 (l),109-114. (25) Hanai, T.; Hatano, H.; Nimura, N.; Kinoshita, T. J. Liq. Chromatogr. 1993,
(19) (20) (21) (22) (23) (24)
16 (4), 801-808.
The pump head was cooled at -5 "C in order to improve its efficiency. The polar modifier was added using a Model 306 pump and mixed with carbon dioxide in a Model 811 dynamic mixer. A Model 831 temperature regulator and a Model 821 pressure regulator provided respectively a temperature and a pressure control. A Model 117 UV variable-wavelength detector equipped with a high-pressure cell was used. Injections were performed using a 231-401 autosampling injector. All results were recorded with a Shimadzu C-R3A integrator. Elemental analyses are consistent with the formula within 3~0.3%(Service Central de Microanalyse du CNRS, Vernaison, France). CSPs were packed into 150 x 4.6 mm i.d. stainless-steel columns by the usual slurry technique under 400 bar using ethanol as pumping solvent. Mobile Phase. For liquid chromatography, ethanol and nhexane were of LiChrosolv grade, purchased from Merck @armstadt, Germany). Chloroform (stabilized with 0.6%(w/w) ethanol) of analytical reagent grade was purchased from Prolabo (Paris). For SFC, carbon dioxide was N-45 grade (99.995%pure, Air Liquide, Alphagaz, Paris). Methanol of LiChrosolv grade was purchased from Merck and n-propylamine (299% pure) from Fluka (Buchs, Switzerland). Solutes. /3-Blockers were purchased from various suppliers. They were used either as tartrate or hydrochloride salts or as free base (the results being the same, because the mobile phase contained n-propylamine and consequently is a basic medium) and simply dissolved in ethanol prior injection. The synthesis of the propranolol analogues has been previously described. Chiral Stationary Phases. CSP 1 (ChyRoSineA) is commercially available from Sedere (Alfortville, France). CSP 2 (improved version of ChyRoSine-A) was synthesized according to ref 26. Both CSPs were based on the same silica (Kromasil, 5 pm, lOO&Eka-Nobel, supplied by Touzart et Matignon). Grafting rates (according to elemental analyses) are equal to 0.25 and 0.24 mmol/g for CSPs 1 and 2, respectively. Molecular Modeling. The molecular modeling software used was SYBYL developed by Tripos associate^.^^ The SYBYL molecular modeling package (version 6.0) runs on a Vax32001 VMS (version 5.42) workstation and operates on the PS300 terminal with the NITRO graphics interface. The molecular mechanics force field implemented in the SYBYL program produces molecular geometries close to those of crystal structures for a selection of various organic molecules.28 The molecules are modeled and minimized (with the molecular mechanics force field) by taking into account their atomic charge distributions, which are calculated by a semiempirical molecular orbital method (MNDO in MOPAC 5 package). The energy m i n i i t i o n procedure (Maximin2) consists of moving the atoms of a molecule in such a way as to generate the atomic coordinates that correspond to a minimum of energy. The energy of the molecule in the force field arises from deviations from "ideal" structural features and can be approximated by a sum of energy contributions such as bending, stretching, torsional, van der Waals and electrostatic contributions, and their cross terms. A system(26) Siret, L.; Tambute, A.; Begos, A; Rouden, J.; Caude, M. Chirality 1991,3, 1-9. (27) Tnpos Associates Inc., 1699 S. Hanley Rd., Suite 303, St. Louis, MO, 63144, SYBYL 5.41, 1988. (28) Clark, M.; Cramer, R D., III; Van Opdenbosch, N.]. Comput. Chem. 1989, 10,982.
Analytical Chemistry, Vol. 67, No. 5, March 1, 7995
953
atic conformational search is used to systematically explore the different viable conformations. Each conformation has a minimized energy value, and the atomic charge distributions are calculated again for the most stable conformations (with low energy value). RESULTS AND DISCUSSION
Chromatographic Results. As it has been previously reported,18 the enantiomeric resolution of p-blockers has been obtained on CSPs 1and 2: baseline resolution is achieved within short analysis time (7 < t < 15 min). CSP 2 displays higher retention (similar grafting rates for CSPs 1and 2) and selectivity values than CSP 1. In this paper, we choose to restrict the study to CSP 2. PropranololAnalogues. In order to get better insights into the chiral recognition mechanism, various propranolol analogues have been studied. It has been previously reported that the steric hindrance displayed by the amine substituent plays an important role in the chiral recognition mechanism. The stereoselectivity is directly connected to its steric bulkiness. Furthermore, as reported in Table 1,the presence of an amine proton is necessary for chiral discrimination to occur, since solute 3 is not resolved and is less retained in SFC. This proton is directly involved in the chiral recognition process, through a stereoselective CSPsolute interaction. In contrast, in liquid chromatography (LC), propranolol (solute 1) and solute 3 exhibit similar k'z values, indicating that this proton is not directly involved in the LC chral recognition mechanism. As reported in Table 1, some other structural changes are not favorable to chral discrimination to occur: the increase of the chain length (solute 2 ) induces a loss of selectivityboth in LC and SFC; the removal of the oxygen linked to the naphthyl ring produces a loss of selectivity whether it is replaced by a methylene or a sulfur group; the replacement of the hydroxyl group by a methoxy group produces a decrease in retention and a strong loss in selectivity both in LC and SFC. Finally, the selectivity is improved by the presence of carbon dioxide only for the solutes having both an amine proton and an ether function, separated one from the other by three carbon atoms. These data allow us to deduce the structural requirements necessary to a good chiral recognition mechanism. These are depicted in Figure 2. Carbon Disulfide. Starting from the previous conclusions that a complexation favorable to chiral recognition occurs between propranolol and carbon dioxide, we try to induce such a complexation with a molecule similar to carbon dioxide, carbon disulfide. In fact, hexane was partly replaced by C% in the liquid mobile phase, and the liquid chromatographic separation of propranolol was conducted in these particular conditions. A differential refractometer detector was used instead of a UV detector due to the high cutoff of C&. No significant improvement of the enantioseparation of propranolol could be obtained using a ternary mobile phase (2%-hexane-ethanol (containing 1%npropylamine). This result indicates that the complexation does not occur with C% as with COZ. The different complexation abilities of COZ and CS, toward propranolol can be explained by the different electronegativities of oxygen (3.50) and sulfur (2.44) and by the induced dipolar moment exhibited by COz and not by CS. Spectroscopic Results. (a) lH NMR. Previous works emphasized that carbon dioxide induces a change in the solute conformation, which suits chiral discrimination. This has been evidenced by NMR experiments. Figure 3 shows the lH NMR 954
Analytical Chemistry, Vol. 67, No. 5, March 7, 7995
Tablo I. Chromatographic Data for tho Soparation of Propranolol (Soluto 1) and Somo Analoguos (Solutos 2-7) on CSP 2 by Llquld Chromatography (LC) and Suporcrltlcal Fluld Chromatography (SFC).
Lc % polar
compounds
k'z
a
5
11.7
1.14
12
19.8 2.07
5
15.5
1
12
12.8 1.07
5
13.2
1
12
11.3 1
2.5
10.7
1
12
10.9 1.07
modifier I
I
SFC % polar
modifier k'z
a
4
5
9.72 1.32
12
24.7 2.27
5
9.2
1
12
13.2 1.08
5 2.5
1.7 2.3
1.01 1.03
12
13.9 1.47
5
6
7
Operating conditions: column, 150 x 4.6 mm id., W detection at 224 mm; LC, mobile phase hexane-ethanol containing 1%(v/v)
n-propylamine, the percentage (v/v) of polar modifier in hexane is indicated in the table; room temperature; flow rate 2 mL/min; SFC, mobile phase COz-methanol containing 1%(v/v) n-propylamine, the percentage (v/v) of polar modifier in COz is indicated in the table; temperature 25 "C; average column pressure 180 bar; flow rate at 0 "C 4 mL/min.
spectrum of propranolol in [2H41methanol (B), the spectrum of the same solution after bubbling of gaseous carbon dioxide (A), and, finally, the spectrum of the same solution after addition of liquid carbon disulfide (C). The change in conformation of propranolol with COz is evidenced by the nonequivalence of protons A The free rotation around the A-B bond is hindered. The chemical shifts of protons C and D move to lower field from Ad = 0.4 and 0.5 ppm, respectively. The chemical shifts and coupling constants of aromatic protons (not represented in Figure 3) are not affected by carbon dioxide; however, the chemical shift of methyl protons (not represented) moves to lower field (Ad =
Ch$n length
bonding
I
Hydrogen bonding
Figure 2. Sites in the fiblocker molecule necessary for chiral discrimination to occur.
Figure 3. ’H NMR spectra in [2H&nethanoi at 360 MHz of propranolol (B), the same solution after bubbling of carbon dioxide (A), and the same solution after addition of carbon disulfide (C).
0.22 ppm). The spectrum obtained after addition of CS, is intermediate between the initial spectrum and the spectrum after bubbling of COZ (for example, the deshielding of protons C and D are respectively A6 = 0.1 and 0.15 ppm), indicating that the influence of CS, is similar in nature but less important than that of COz. This observation may explain why no significant improve ment of the selectivity could be obtained using CS, as additive in the liquid mobile phase. The slight selectivity improvement is perhaps not observed due to the low sensitivity of the differential refractometer detector (the column capacity was reached). Similar results are obtained for solute 5, indicating that the nature of the amidic alkyl substituent has no influence in the interaction with COZ. No modification of the NMR spectrum occurs after bubbling of COZfor solutes 2-4,6, and 7, indicating that the sites described in Figure 2 are necessary to the complexation. (b) Infrared Spectroscopy. This method does not allow us to show the complexation of propranolol by COZ,the adsorption bands supposedly involved in the complexation (i.e., NH and OH bands) being too weak. Molecular Modeling. The purpose of any molecular modeling program is to build, to study, and to manipulate molecules. It
is an aid to give the shapes of the minimum energy conformation and to calculate the charge distribution. The chiral stationary phase and the propranolol molecule have been modeled separately. The considered conformations were selected from the most stable conformations (with low minimized energy) and in agreement with information given by the ‘H NMR spectra. For example, the presence of hydrogen bonding in the conformation induces a hindrance of free rotation around some bonds; hydrogen atoms of CHZcarrying different charges are not equivalent in ‘H NMR, and inversely, hydrogen atoms of CHZcarrying the same charges are equivalent. Previous works emphasized that the CSP conformation is not modified by supercriticalcarbon dioxide. The considered CSP conformations were therefore the same with and without carbon dioxide. However, carbon dioxide induces a change in the solute conformation which was evidenced by NMR experiments as described above. All this information allowed us to choose the right conformationsof solvated propranolol that may exist with and without carbon dioxide. Then, the selected conformations of each propranolol enantiomer were made to approach the conformation of CSP 2. Each association was minimized and characterized by some specific interactions and a minimized energy value. At last, only one conformation of propranolol accounted for the results of the SFC enantiomeric separation. The obtained model is also j u s ~ e by d considering the data concerning the propranolol analogues. Modeling of CSP 2. The optimized structure of CSP 2 is presented in Figure 4. Only the CS (with the spacer) was modeled, and the silica by itself was schematized by a hatching wall. The minimized structure is represented from two viewing directions. The conformation of (R)-CSP 2 is similar to the previous (S)-CSP 2 conformation. They also exhibit close energy values: E((S)-CSP 2) = E((R)-CSP2 = -18 f 0.1 kcal/mol. These conformationsare not modified by the presence of carbon dioxide. The overall shape is bent. A linear chain is directly bound to the silica. The bend is caused by the benzene ring. The functional groups, located on each side of the chral carbon atom, constitute the active part of the CSP, placed perpendicularly to the linear chain and parallel to the silica surface. The optimized structure shows the existence of an intramolecular hydrogen bonding between the oxygen atom of the carbonyl group and the hydrogen atom of the amido group, which are close to the chiral center. This peculiar structure forms a cavity (indicated by an arrow in Figure 4, view b) in which are located three accessible interaction sites: 3,5-dinitrobenzoyl group, amino group, and carbonyl group. We should expect that the solute could enter into this cavity and form with the CSP as an “inclusion complex”. The 3,5-dinitrobenzoyl group is a nacid group which may be involved in a charge transfer complex formation with solutes possessing a complementary z-basic group, spatially locked in close parallel planes. The amido group has an acidic hydrogen atom (partial charge +0.221) and the carbonyl group has a basic oxygen atom (partial charge -0.376) which may be involved in hydrogen bondings with solutes possessing respectively basic oxygen and acidic hydrogen. These two groups are close to the chiral center, and their interactions with the solutes may be necessary for chiral recognition to occur. Modeling of (R)and (8-Propranolol. As described above, the change in conformation of propranolol in the presence of carbon dioxide was evidenced by ‘H NMR A systematic conformational study allows us to generate low-energy conformations. Analytical Chemistty, Vol. 67, No. 5, March 7, 7995
955
b)
SILICA
cavit v
SILICA
Figure 4. Optimized structure of CSP 2 from two viewing positions.
The selected conformations should agree with the conditions obtained from the corresponding lH NMR spectra. The two main conditions are, first, the presence of an intramolecular hydrogen bonding which induces a hindrance of free rotation around the A-B bond and, second, the atomic charges carried by protons A. The conformations without carbon dioxide are characterized by equivalent protons A, carrying the same charge when those of conformationsin the presence of carbon dioxide are nonequivalent and do not carry the same charge. The selected conformations of propranolol that allow us to account for the results of enantiomericseparation using LC ((S)confl and (I?)confl without C02) and SFC ( ( 9 - c o d and (R)-co& with C02) are given in parts a and b of Figure 5, respectively. Atomic charge values have been calculated for protons A: they are equivalent without C02 (0.011 and 0.010) and different with C02 (0.012 and 0.006). This indicates that the proposed structures are in good agreement with the lH NMR spectra of propranolol without and with carbon dioxide. As expected, for a given conformation, the enantiomers exhibit close minimized energy values: E ( R ) -=~E($cod ~ ~ I = 6.57 f 0.05 kcal/mol and E(m-cona = Eg-conE = 8.7 f 0.01 kcal/mol. The proposed conformationwithout C02 exhibits an intramolecular hydrogen bonding between the hydrogen atom of the amino group and the oxygen atom of the ether function, giving rise to a very stable six-memberedring. In the presence of carbon dioxide, this hydrogen bond is broken due to a specific solvation of both the amino and the hydroxyl functions. The selected stable conformations, (R)eonf2and (S)-conf2, have no more cyclic form but still present two intramolecular hydrogen bonds as depicted in Figure 5b. The change in conformation of propranolol due to the presence of carbon dioxide is mainly induced by a rotation around the A-B bond. The structures of (R)-confZ and (S)-conf2 show three potential interaction sites (naphthyl ring, ether and hydroxyl functions), simultaneously accessible on the same side of the conformation. In order to check that the modeled conformations are in good agreement with the NMR experiments, the angles deduced from the coupling constants (via the Karplus relation) are compared to the angles measured in SYI3YL. These data are gathered in Table 2. 956 Analytical Chemistry, Vol. 67, No. 5, March 1, 1995
a) (R)-propranolol
b) (R)-propranolol, n C 0 2
A
B
Figure 5. (a) Optimized structure of (/?)-propranolol without CO:! labelled (R)-confl . The intramolecular hydrogen bonding is schematized by an arrow. (b) Optimized structure of (R)-propranolol with CO2 labelled (m-conf2. In order to simplify the figure, only two molecules of carbon dioxide are schematized.
Modeled Propranolol-CSP 2 Associations. The (I?)propranolol- (s)-CSP 2 and (3-propranolol- (3-CSP 2 associations are built with the modeled conformation of (3-CSP 2 and the conformations of propranolol (I?)-confl, (3-confl (LC) and (R)eo&, (s)-conf2 (SFC with C02). The (I?)-confl- (5')-CSP 2 association is represented in Figure 6. There are two interactions. The first main interaction is the
The (R)-propranolol- (9-CSP 2 and (3-propranolol- O C S P 2 associations with carbon dioxide are represented respectively in Figures 7 and 8. In the case of (R)-conf2- (3-CSP 2 association (Figure 7), the (@coni2 is located inside the cavity of the CSP and exhibits three strong interactions with CSP one n-n interaction and two hydrogen bondings (1 and 2, Figure 7), one between the ether function and hydrogen atom of the amido group of CSP 2, and the other between the hydrogen atom of the hydroxyl group and the oxygen atom of the carbonyl group of CSP 2. The isopropyl group is located on the outside of the cavity and does not cause steric hindrance for the approach. Two other lower hydrogenbonding interactions (3 and 4,Figure 7) may also occur: the one between the amine proton of 0-propranolol and the oxygen atom of the carbonyl group of CSP 2 and the other between the oxygen atom of the hydroxyl function and the amine proton of CSP 2. All the measured distances of the hydrogen bonds are indicated in Table 3. So the (@-coni2 conformation of (R)-propranolol gives stronger interactions with (3-CSP 2. In the case of Qconf2- (3-CSP 2 association (Figure 8), only one interaction is possible: it is the n-n interaction. All the measured distances between the atoms opening to hydrogen-bonding interactions are too great. They are indicated in Table 3. This fact is due to the presence of isopropyl group located on the inside of the cavity. In this case, the isopropyl group induces a steric hindrance that is unfavorable to the formation of strong hydrogen-bondinginteractions. Then @-conf2 is much less fixed on (3-CSP 2 than (R)-conf2. So, molecular modeling allows us to account for the chiral recognition of propranolol by CSP 2 and the good selectivity of the enantiomeric separation. Propranolol Analogues. The proposed model has been tested by taking into account the above described chromatographic data involving propranolol analogues. The modscation of different parts of the solute is considered ether function, amine proton, hydroxyl function, amine alkyl substituents, influence of n-basic group,and solute 2. These results will be published later.
Table 2. Dihedral Angle Values Obtained by NMR (Flgure 1) Accordlng to the Karplus Equation and by Molecular Modellng for the Propranolol in the Presence (conf2, Figure 3b) or Absence (confl, Flgure 3a) of Carbon Dioxide
dihedral angle Ha-Hc
NMR SYBYL
Hb-Hc
Hd-Hc
(R) 177.7 (S)177.8
(R) 63.3
64.3
He-Hc
56.9
(R) 59.1
(S)59.2
156.6
(R) 179
(3 179
(S)63.3
r
_1
HaHc Hd H CH3 O W i d + H ; n(O=C=O) HbOH He CH3
dihedral angle Ha-Hc
NMR SYBYL
Hb-Hc
Hd-Hc 59.6
169.4
(R) 60.7 (S)60.5
(R) 61.8 (S)61.0
(R) 179.4
63.6
(R) 58.5
(S)58.7
He-Hc
(S)179.7
n-n interaction between the 3,5dinitrobenzoylgroup of CSP 2 and the naphthyl ring of propranolol, which are spatially locked in close parallel planes. The distance measured between the two planes is about 3 A. The second interaction is hydrogen bonding between the oxygen atom of the nitro group of CSP 2 and the amine proton of propranolol. In this case, there is no interaction between CSP and the hydroxyl function carried by the chiral center, but we can imagine a nonstereoselectivehydrogen bond interaction between the hydroxyl function and the silica. In the (S)-confl- QCSP 2 association, two similar interactions occur. So, following this molecular modeling, two interactions are involved whatever the enantiomer. The chiral recognition of propranolol by CSP is practically not possible and then the selectivity of the enantiomeric separation is close to 1.
CONCLUSION Molecular modeling coupled with lH NMR has allowed us to propose a model to explain the difference in results obtained on the enantiomeric separation of propranolol on (9-ChyRoSine-A and its improved version by SFC (with carbon dioxide) and by
Table 3. Measured Interaction Distances (In A) between (R). and (S)-Propranolol and (S)-CSP 2 with and wlthout Carbon Dioxide NOIIIZ
CSP 2
I
Propranolol
t Interactions 2
(a)
3
4
n-n
Oi-Hn
Hio-01
Hi4-0~
Hi4-01
02-H11
3 3
2.70 4.52
1.92 6.89
3.62 6.22
3.47 6.30
1
with C02 (SFC) (R)-conn- (3-CSP 2 (S)COnn-(S)-CSP 2 without con (Lc) (R)-confl-(s)-CSP 2 (s)confl-Q-CSP 2
3 3
2.00 1.89
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SILICA Figure 6. Optimized association between the optimized structures of (R)-propranolol (without C02) and CSP 2 ((R)-confl-(S)-CSP 2).
SILICA Figure 8. Optimized association between the optimized structures of (S)-propranolol with CO2 and CSP 2 ((S)-confP-(S)-CSP 2).
cannot develop stereoselective interactions with the CSP and the interactions involved are the same whatever the enantiomer. The solvating effect of carbon dioxide induces a change of propranolol conformations that are geometrically favorable to the chiral recognition process. In this case, the conformation of (R)propranolol involves more interactions with (8-CSP than the conformation of (8-propranolol. The validity of this model is tested by other chromatographic data obtained with various ,%blockersand propranolol analogues. We note that the change of conformation of the analogue solutes occurs in the presence of carbon dioxide when the solutes have both an amino proton and an ether function separated one from the other by three carbon atoms. The advocated model is presented with (8-CSP 2 but it could be presented in the same way with the corresponding (R)-CSP. In this case, all the results should be inversed. SILICA Figure 7. Optimized association between the optimized structures of (R)-propranolol with COn and CSP 2 ((R)-conf2-(S)-CSP 2).
LC. In this work, we visualize the conformations of the chiral phase, (a-solute, (S)-solute,and their respective associations.The solute conformations are selected by taking into account the information given by the ‘HNMR spectra. Without carbon dioxide, the selected conformationsof (R)-and (3-propranolol have such geometrical structures that the chiral recognition process is not possible: the chiral center of the solute
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ACKNOWLEWMENT The authors express their grateful thanks to F. Vergne (Centre #Etudes du Bouchet) for the synthesis of propranolol analogues and to D. Loeillet (Centre #Etudes du Bouchet) for NMR experiments. Received for review March 15, 1994. Accepted October 13, 1994.@ AC940261 P Abstract published in Adoance ACS Abstracts, December 1, 1994.
